Treatment of inflammatory bowel disease

ABSTRACT

Patients suffering from inflammatory bowel disease such as Crohn&#39;s disease or ulcerative colitis are treated either orally or intravenously with methylol transfer agents, such as taurolidine and/or taurultam. These agents can be used in combination with other drugs thereby allowing the use of smaller amounts of the other drugs and limiting unwanted side effects.

BACKGROUND OF THE INVENTION

[0001] Inflammatory bowel disease (IBD) is of unknown etiology, althoughimmunological mechanisms play a significant role. The two majordisorders involved are ulcerative colitis and Crohn's disease. Bothdiseases are chronic relapsing disorders.

[0002] The exact pathogenesis of IBD is unknown. Various factors such asenvironmental, genetic, smoking and infectious agents have beensuggested.

[0003] IBD is characterized by a chronic remitting and relapsing course.The general aims of treatment are to induce remission and preventrelapse. The approach to therapy varies according to type, distributionand severity of disease in individual patients.

[0004] Current therapies for IBD include anti-inflammatories andsteroids and sulphasalazine.

[0005] Immunosuppressive agents such as azathioprine, 6-mercaptopurine,cyclosporin and methotrexate are emerging as potentially useful agentsin severe and refractory cases of IBD.

[0006] Other treatment modalities undergoing investigation includelipoxygenase inhibitors, fish oil and hydroxychloroquinine.

[0007] Exacerbation of inflammatory bowel disease, ulcerative colitisand Crohn's disease, is marked by local release of proinflammatorymediators, increased vascular permeability, and recruitment of acuteinflammatory cells, which ultimately leads to mucosal ulceration. It hasbeen shown that proinflammatory cytokines such as tumor necrosisfactor-a (TNF-α), interleukin-1β, (IL-1β) and interleukin-6 (IL-6) tendto be consistently elevated in patients with active IBD. Furthermore,increased levels of angiogenic cytokines such as vascular endothelialgrowth factor (VEGF) has been recently demonstrated in patients withIBD. Peripheral monocytes and intestinal macrophages from patients withIBD have been found with an enhanced ability to secrete increasedamounts of proinflammatory cytokines. An increased capacity to secreteIL-1β and TNF-α, two proinflammatory cytokines particularly importantfor inducing and sustaining intestinal inflammation in IBD, has beenfound in polymorphonuclear neutrophil granulocytes (PMN) from patientswith active IBD. Reactive oxygen species and nitric oxide mainlyreleased from PMN in patients with IBD have been considered as importantfactors in the pathogenesis of IBD. It is now becoming clear thatinflammatory cells and monocyte- and PMN-released mediators may play akey role in the amplification of inflammation and tissue damage in IBD.

[0008] Lipopolysaccharide (LPS) or endotoxin comprises the key elementof most gram-negative and some gram-positive bacteria. LPS is animportant mediator of gram-negative sepsis and septic shock. LPS itselfhas been implicated as one of the potent inducers of proinflammatorymediator synthesis and release, including cytokines and reactive oxygenmetabolites in inflammatory cells. Endotoxemia may occur in the absenceof gram-negative bacteremia because endotoxin can transit the normal gutwall in small amounts. Such transiting of endotoxin is potentiallyincreased by the presence of mucosal inflammation in patients withactive IBD, which eventually leads to endotoxemia.

SUMMARY OF THE INVENTION

[0009] In accordance with the present invention, a method of treatinginflammatory bowel disease in a patient comprises administering to thepatient an effective amount of a methylol transfer agent.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Without being bound to any particular theory, it is believed thatblock of endotoxin both systematically and locally may have beneficialeffect on IBD through inhibition of inflammatory cell activation andreduction of proinflammatory mediator release. Taurolidine is a provenchemotherapeutic agent with a potent bactericidal and antiendotoxineffect. Its mechanism of action, unlike that of antibiotics, is based ona chemical reaction. During the metabolism of taurolidine to taurinamideand ultimately taurine and water, methylol groups are liberated andchemically react with the mureins in the bacterial cell wall and theamino and hydroxyl groups of endotoxin and exotoxins. This results indenaturing of the complex polysaccharide and lipopolysaccharidecomponents of the bacterial cell wall and endotoxin and in theinactivation of susceptible exotoxins.

[0011] The present invention is applicable to any suitable methyloltransfer agent that reduces inflammatory bowel disease in a patient.Although the invention is further described with respect to the methyloltransfer agents taurolidine and/or taurultam, it is to be understoodthat the invention is equally applicable to any suitable methyloltransfer agent having activity similar to or substantially the same astaurolidine and/or taurultam.

[0012] Methylol transfer agents in accordance with the present inventioncan be administered in any suitable form, such as orally administeredtablets or capsules, or intravenously 30 administered solutions.

[0013] Taurolidine (bis(1,1-dioxoperhydro-1,2,4-thiadiazin-4-yl)methane)has been employed as a clinically effective therapeutic agent for manyyears. The compounds taurolidine and taurultam are as disclosed in U.S.Pat. No. 5,210,083, incorporated herein by reference.

[0014] Taurolidine has been utilized both for antibacterial prophylaxisand as a therapeutic bactericidal agent in peritoneal sepsis. It has ashort half life and is rapidly metabolized to taurine, carbon dioxideand water. Taurolidine has been shown to have a broad spectrum ofantimicrobial activity against both gram positive and gram negativebacteria and fungi and has a neutralizing activity against bacterialendotoxin. Taurolidine has been shown to be non-toxic to humans andanimals and is free from side effects following intravenous andintraperitoneal administration.

[0015] This wide spectrum of antiseptic properties has led to itsclinical application in conditions ranging from osteomyelitis toperitonitis and catheter related sepsis prophylaxis.

[0016] The use of methylol transfer agents in IBD may be based on theirspecific modes of action. These include: 1) Reduction/Inhibition of theinflammatory reaction, 2) Selective destruction of pathogenic bacteria,and 3) Protection of epithelial cells in the gut wall.

[0017] Preferred dosages contain about 100-1000 mg taurolidine and/ortaurultam, most preferably about 200-500 mg thereof. Dosages may beadministered 1, 2, 3, 4, 5 or more times per day, preferably on a dailybasis.

[0018] Administration of taurolidine together with known IBD treatmentagents could allow the use of lower amounts of the other agents, e.g.,using taurolidine in combination with Remicade for treating Crohn'sdisease could allow the use of lower amounts of Remicade. The use oftaurolidine to decrease the necessary levels of other drugs willdecrease any deleterious side effects which may be associated with useof the higher levels of those other agents.

[0019] The present invention may be applicable, inter alia, to: A)Patients with Crohn's disease active and inactive the effectiveness ofwhich can be measured by using the Crohn's disease activity index(CDAI); and B) Patients with ulcerative colitis active and inactive, theeffectiveness of which can be measured by using the clinical colitisactivity index (CAI).

[0020] According to one embodiment, patients receive a single oral doseof 20 g taurolidine suspension per day for ten days.

[0021] The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1

[0022] This experiment evaluates the beneficial effects of oraladministration of taurolidine on attenuation of systemic inflammatoryresponse in patients with active inflammatory bowel disease. ThisExample measures systemic proinflammatory and angiogenic cytokines,systemic reactive oxygen species and nitric oxide in patients with IBDpre- and post-taurolidine administration. Secondly, circulating PMN andmonocytes from patients with IBD are assessed for their adhesionreceptor expression, and their ability to release proinflammatory andangiogenic cytokines, as well as reactive oxygen species and nitricoxide following taurolidine treatment.

[0023] A) Experimental Design

[0024] 1) Peripheral venous blood samples are collected from patients onthe day before taurolidine administration, and day 2, day 5 and day 10after taurolidine administration. Serum samples are harvested bycentrifugation and stored at −80° C. for measurement of serum levels ofendotoxin, LPS binding protein (LBP), and soluble CD14, serumproinflammatory cytokines (TNF-α, IL-1β, IL-6), serum angiogeniccytokines (VEGF, TGF-β1), serum lipid peroxides (malonaldehyde), nitricoxide and peroxynitrite.

[0025] 2) Receptor expression of CD11a, CD11b, CD18, and CD14 on PMN andmonocytes in the whole blood samples are assessed on the day beforetaurolidine administration, and day 2, day 5, and day 10 aftertaurolidine administration. PMN respiratory burst and phagocytosis inthe whole blood samples are examined at different time points pre- andpost-taurolidine administration.

[0026] 3) Circulating PMN and monocytes are isolated from patients onthe day before taurolidine administration, and day 5 and day 10 aftertaurolidine administration using the Dextran-Ficoll density gradienttechnique. Isolated PMN and monocytes will be assessed for theirspontaneous and LPS-stimulated proinflammatory cytokine release and forangiogenic cytokine release.

[0027] 4) Clinical symptoms and signs in patients with IBD are examinedon the day before taurolidine administration, and day 5 and day 10 aftertaurolidine administration.

[0028] B) Methodology

[0029] Dextran-Ficoll gradient sedimentation for circulating PMNisolation; solid-phase ELISA for assessment of proinflammatorycytokines, angiogenic cytokines, serum LBP and soluble CD14; flowcytometry for determination of PMN and monocyte receptor expression,respiratory burst and phagocytosis; Limulus amebocyte lysate assay fordetection of serum endotoxin levels; colorimetric assay fordetermination of lipid peroxidation; fluorescent probe DHR 123 andGreiss reaction for measurement of peroxynitrite and nitric oxide, areutilized respectively.

[0030] C) Results

[0031] There are increased systemic proinflammatory and angiogeniccytokine levels, and increased PMN and monocyte adhesion receptorexpression in patients with active IBD. Systemic lipid peroxides, nitricoxide and peroxynitrite also are increased with enhanced potential ofPMN and monocytes to release proinflammatory and angiogenic cytokines,as well as reactive oxygen species in patients with active IBD.Administration of taurolidine ameliorates these phenomena in patientswith IBD.

EXAMPLE 2

[0032] This example evaluates the beneficial effect of oraladministration of taurolidine on amelioration of inflammatory responsein intestinal mucosa in patients with active inflammatory bowel disease.This Example measures proinflammatory cytokines, angiogenic cytokines,reactive oxygen species and nitric oxide in the mucosal biopsy culturesin patients with IBD pre- and post-taurolidine administration. Localmucosal recruited PMN and mononuclear leukocyte-associatedproinflammatory cytokine and angiogenic cytokine expression in patientswith IBD are assessed pre- and post-taurolidine treatment.

[0033] A) Experimental Design

[0034] 1) The specimens of standard intestinal mucosal biopsy arecollected from patients with IBD on the day before taurolidineadministration, and day 5 and day 10 after taurolidine administration,and incubated with completed culture medium for different time points.The supernatants from the biopsy cultures are harvested bycentrifugation and stored at −80° C. for measurement of proinflammatorycytokines (TNF-α, IL-1β, IL-6), angiogenic cytokines (VEGF, TGF-β1),lipid peroxides (malonaldehyde), nitric oxide and peroxynitrite.

[0035] 2) The mononuclear leukocytes from the above specimens of mucosalbiopsy are isolated using the technique of collagenase digestion anddensity gradient sedimentation. TNF-β, IL-1β, IL-6 and VEGF protein andmRNA expression in these mononuclear leukocytes are assessed.

[0036] 3) The specimens of standard intestinal mucosal biopsy frompatients with IBD on the day before taurolidine administration, and day5 and day 10 after taurolidine administration are assessed for PMN- andmonocyte-associated TNF-a, IL-1, IL-6 and VEGF expression.

[0037] 4) Pathological changes of intestinal mucosa in patients with IBDare examined on the day before taurolidine administration, and day 5 andday 10 after taurolidine administration.

[0038] B) Methodology

[0039] Collagenase digestion and Percol gradient sedimentation forisolation of mucosa-associated mononuclear leukocytes; solid-phase ELISAfor assessment of TNF-α, IL-1β, IL-6, VEGF and TGF-β1 in supernatantsfrom the biopsy cultures; flow cytometry and RT-PCR for determination ofintracellular TNF-β, IL-1β, IL-6 and VEGF protein expression and mRNAexpression in mucosa-associated mononuclear leukocytes, respectively;colorimetric assay for determination of lipid peroxidation; fluorescentprobe DHR 123 and Greiss reaction for measurement of peroxynitrite andnitric oxide, respectively; immunocytochemistry for detection of PMN-and monocyte-associated TNF-α, IL-1 β, IL-6 and VEGF expression inmucosal biopsy specimens, are utilized.

[0040] C) Results

[0041] Patients with active Crohn's disease and active ulcerativecolitis have increased levels of proinflammatory cytokines, angiogeniccytokines, and reactive oxygen species, which is related to theincreased recruited PMN and monocytes in intestinal mucosa. Treatmentwith taurolidine improves pathological changes in intestinal mucosa inpatients with active IBD, which is associated with downregulation ofproinflammatory cytokines, angiogenic cytokines, reactive oxygenspecies, and recruitment of PMN and mononuclear leukocytes in theintestinal mucosa by taurolidine.

[0042] The above Examples are repeated using intravenous administrationof taurolidine.

[0043] Examples of the tablets to be utilized or the intravenousformulations to be used are described in the following Examples.

EXAMPLE 3 Tablets with Gastric Juice Resistant Coating Soluble in theBowel

[0044] Tablets comprising 200 mg taurolidine with a total tablet weightof approximately 460 mg, a diameter of 11 mm and a thickness of 4 mmwere prepared. Each tablet comprises: Com- ponent Name Amount 1Taurolidine 200 mg 2 Emdex ® 100 mg (Penwest Pharm. co., NY, USA)(Dextrates, NF hydrated) 3 Starch 1500 100 mg 4 Talcum  8 mg 5Mg-stearate  1 mg 6 Aerosil 200  1 mg

[0045] To manufacture the tablets, the components are mixed in astainless steel container and rotated on a Rhoen-wheel or in a stainlesssteel mixing machine. Components 1-3 are preliminarily mixed forapproximately 10 minutes. Thereafter, components 4-6 are added and mixedfor another 10 minutes. Formation of the tablets is on anexcenter-tablet press.

[0046] Coating of the tablets is performed in a coating pan with agastric juice resistant coat on 30 the basis of an acrylic resin, usingthe Eudragit® types of Rhon Pharma GmbH, D-Darmstadt, Germany. Coatingis effected under slow rotation with a spray system using a solution ofEudragit® L30D (Poly[meth]acrylic acid ester, MW 800,000) in anisopropyl-aicohol:water mixture of 70:30. Alternatively, Eudragit® ELmay be used for the coating.

[0047] Varnishing is effected by a 12.5% solution in isopropyl-alcohol.Thereafter the coated/varnished tablets are dried in a vacuum-dryingoven.

EXAMPLE 4 Tablets with Gastric Juice Resistant Coating Soluble in theBowel

[0048] Tablets are prepared as in Example 3 except 200 mg taurultam areused in place of the 200 mg taurolidine.

EXAMPLE 5 Tablets with Gastric Juice Resistant Coating Soluble in theBowel

[0049] Tablets are prepared as in Example 3 except that in place of 200mg taurolidine, the tablets comprise 100 mg taurolidine and 100 mgtaurultam.

EXAMPLE 6 Slow Intravenous Drop Infusion-I.V. Infusion Solution forCrohn's Disease

[0050] An intravenous solution for administration via a central catheteror port is prepared in volumes of 100, 250 or 500 mL with taurolidine at2% in glass bottles of 100, 250 or 500 mL with rubber stoppers andaluminum caps. Component Name Amount 1 Taurolidine 1 g 2 Taurultam 1 g 3Povidone UP 5 g (see U.S. Pat. No. 6,080,397) 4 Sterile water to 100 mLpH after sterilization is 7.2-7.3.

EXAMPLE 7 Slow Intravenous Drop Infusion-I.V. Infusion Solution forCrohn's Disease

[0051] An intravenous solution for administration via central catheteror port is prepared as in Example 6 but with the following: ComponentName Amount 1 Taurolidine 20 g 2 Povidone UP 50 g (see U.S. Pat. No.6,080,397) 3 D-glucosemonohydrate 10 g 4 Sterile water to 1000 mL pHafter sterilization is 6.8.

EXAMPLE 8 Administration of Taurolidine to Patients with InflammatoryBowel Disease

[0052] Four patients with inflammatory bowel disease were treated dailywith orally administered 300 mg taurolidine capsules.

[0053] While the invention has been disclosed by reference to thedetails of preferred embodiments of the invention, it is to beunderstood that the disclosure is intended in an illustrative ratherthan in a limiting sense, as it is contemplated that modifications willreadily occur to those skilled in the art, within the spirit of theinvention and the scope of the appended claims.

What is claimed is:
 1. A method of treating inflammatory bowel diseasein a patient comprising administering to said patient an effectiveamount of a methylol transfer agent so as to reduce systems ofinflammatory bowel disease in said patient.
 2. The method of claim 1wherein said agent is selected from the group consisting of taurolidine,taurultam and mixtures thereof.
 3. The method of claim 1 wherein saidagent is orally administered to said patient.
 4. The method of claim 3wherein taurolidine is administered to said patient at an oral dose ofabout 100-1000 mg.
 5. The method of claim 4 wherein said taurolidine isadministered to said patient daily.
 6. The method of claim 5 whereinabout 20 g of said taurolidine is administered to said patient daily. 7.The method of claim 1 wherein said agent is administered intravenouslyto said patient.
 8. The method of claim 1 wherein said inflammatorybowel disease is Crohn's disease.
 9. The method of claim 1 wherein saidinflammatory bowel disease is ulcerative colitis.
 10. The method ofclaim 1 wherein a drug in addition to said methylol transfer agent isadministered to said patient.